(433b) Binding Free Energy Calculation on Human Beta Defensin Type 1 (hBD-1) and Type 3 (hBD-3) on Bacterial Membrane

Human beta defensins (hBD) are small antimicrobial peptides acting as an important part of human innate immunity. They have antimicrobial activities against different microorganisms including bacteria, virus, fungi. Several human beta defensins have been isolated so far and all of them have the ability to kill or inhibit bacteria at low salt concentrations. It is believed that hBDs interacts with bacteria by disrupting its membrane. So, it is important to investigate its interaction with the bacterial membrane in molecular details to understand the antimicrobial mechanism. Among the hBDs, hBD-3 is the most potent and found to be active against both Gram-positive and Gram-negative bacteria even at high salt concentrations. It has 45 residues and a charge of +11. hBD-1 has 36 residues and a charge density of +7. hBD-1 shows antimicrobial activity against some gram-negative bacteria at low salt concentration, but relatively less potent against gram-positive bacteria. In this study, we are going to investigate the binding free energy of the residues on hBD-3 and hBD-1, with a model bacterial membrane consisting of pure POPG lipids; because both Gram-positive and Gram-negative bacterial membranes consist of POPG lipids at high concentrations, 67% and 33% of the total phospholipids respectively. Comparing the binding free energy results of hBD-3 with hBD-1 might give an insight of the residues which make hBD-3 potent against both gram-positive and gram-negative bacteria. As it is quite complicated to calculate the absolute binding free energy of the residue with membrane, the relative binding free energy of protein will be calculated resulting from the point mutation of each residue to Alanine. A thermodynamic cycle has been designed to calculate the difference in the binding free energy. The alchemical free energy changes will be calculated by running two free energy perturbation simulations of the mutation and the difference between the mutation free energy gives the difference in the binding free energy of the protein in wildtype and the mutant. The simulations have been performed using NAMD program at a temperature of 300.15 K and a pressure of 1 atm, dividing the whole process into 20 different thermodynamic windows and running each window for 3ns in the bound state simulation and 2ns for unbound state simulation. Based on the free energy results of hBD-3, it is found that Arginine 17, Arginine 36 and Arginine 43 bind quite strongly with the POPG lipid compared to the other residues contributing to a total binding free energy of –9.1297 kcal/mol out of -28.388 kcal/mol. The work emphasized the contribution of positively charged Arginine residues to the binding between hBD-3 and bacterial membrane.